Novel tracers for in vivo studies of waste transport by fluid flows in the brain

用于脑内液体流动废物运输体内研究的新型示踪剂

• 批准号: 10732612
• 负责人: Nozomi Nishimura
• 金额: $ 44.71万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732612
• 关键词: APP-PS1; Abeta clearance; Acute; Address; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein; Animals; Area; Basement membrane; Belief; Biological Assay; Blood Vessels; Brain; Cells; Color; Control Animal; Convection; Dementia; Deposition; Development; Dextrans; Disease; Drainage procedure; Dyes; Exposure to; Extracellular Protein; Failure; Gene Activation; Genes; Genetic Engineering; Hour; Injections; Intercellular Fluid; Lighting; Link; Liquid substance; Location; LoxP-flanked allele; Lymphatic; Maps; Measurement; Measures; Mediating; Meningeal lymphatic system; Methods; Microscopic; Modeling; Morphologic artifacts; Motion; Mus; Neurons; Peptides; Permeability; Process; Production; Protein Secretion; Proteins; Regulation; Reporter; Reporter Genes; Reporting; Resolution; Route; Speed; System; Testing; Therapeutic; Time; Tissues; Tracer; Travel; Variant; Viral Vector; abeta accumulation; adeno-associated viral vector; aged; arteriole; brain parenchyma; brain tissue; brain volume; extracellular; fluid flow; fluorescence imaging; fluorophore; glymphatic system; improved; in vivo; interstitial; intravital imaging; lymph nodes; lymphatic circulation; lymphatic vessel; mouse model; novel; pressure; prevent; protein aggregation; protein transport; prototype; temporal measurement; theories; tool; venule; wasting; APP-PS1; Abeta clearance; Acute; Address; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein; Animals; Area; Basement membrane; Belief; Biological Assay; Blood Vessels; Brain; Cells; Color; Control Animal; Convection; Dementia; Deposition; Development; Dextrans; Disease; Drainage procedure; Dyes; Exposure to; Extracellular Protein; Failure; Gene Activation; Genes; Genetic Engineering; Hour; Injections; Intercellular Fluid; Lighting; Link; Liquid substance; Location; LoxP-flanked allele; Lymphatic; Maps; Measurement; Measures; Mediating; Meningeal lymphatic system; Methods; Microscopic; Modeling; Morphologic artifacts; Motion; Mus; Neurons; Peptides; Permeability; Process; Production; Protein Secretion; Proteins; Regulation; Reporter; Reporter Genes; Reporting; Resolution; Route; Speed; System; Testing; Therapeutic; Time; Tissues; Tracer; Travel; Variant; Viral Vector; abeta accumulation; adeno-associated viral vector; aged; arteriole; brain parenchyma; brain tissue; brain volume; extracellular; fluid flow; fluorescence imaging; fluorophore; glymphatic system; improved; in vivo; interstitial; intravital imaging; lymph nodes; lymphatic circulation; lymphatic vessel; mouse model; novel; pressure; prevent; protein aggregation; protein transport; prototype; temporal measurement; theories; tool; venule; wasting

SUMMARY Interstitial fluid motion in the form of convective flow is hypothesized to contribute to amyloid beta (Aβ) and waste clearance by carrying extracellular proteins out of the brain. Failures of this system could contribute to Alzheimer’s disease (AD), but are some aspects of this theory are still speculative. For examples, while the inflow of fluid to the brain parenchyma is well documented, the outflow path is less clear. How exogenous tracers and brain proteins get from the tissue into the lymphatics is still an open question. There are at least two ideas. The glymphatic/lymphatic transport mechanism predicts that the inflow of fluid from the arteriole perivascular spaces would be balanced by an outflow through the venular perivascular spaces. The Intermural Peri-Arterial Drainage (IPAD) camp has identified that extracellular dyes and proteins aggregate near the basement membrane of the arteriole vessels and hypothesizes that this compartment provides a conduit for waste clearance. The IPAD idea predicts that waste exits along the arterioles, in the opposite direction of the glymphatic prediction, although both are thought to feed into the lymphatics. There are experimental challenges in studying this transport because fluid motion is usually tracked by injection of exogenous indicators that alter pressure balances and can only be used for acute measurements. To overcome these barriers, new genetically engineered secreted tracers (GESTs) which mimic endogenous protein production and are not complicated by artifacts from injection have been prototyped. Microinjected adeno-associated virus (AAV) vectors drive the expression of GESTs in neurons within a small volume of brain. The distribution of the secreted fluorescent protein using fluorescence imaging in both tissue sections and intravital imaging can be used to map the transport. Because this tracer is produced by neurons long after the injection of the viral vectors, pressures and fluid flow in the brain are not disturbed. In addition, proteins such as Aβ are also secreted by neurons, so that the GEST distribution will replicate native protein transport rather than the motion of exogenously injected tracers. While this strategy has shown promise, new capabilities are needed to address the specific questions about fluid transport of waste proteins. First, interstitial fluid flow rates in the brain require faster time resolution on the scale of minutes. A photoactivable version of the GEST is proposed so that a volume of tracer can be “highlighted” and tracked as it moves through the brain tissue. The direction and speed of motion could support or refute the different theories of flow-mediated waste clearance. Second, it is not possible to detect GESTs in areas of faster flow or low concentration such as the lymphatics. To detect the paths taken by secreted proteins, a new secreted, cell-permeant Cre is developed. This will be used in floxed-stopped reporter animals to turn on reporter genes in cells that encounter fluids carrying the Cre. Because the gene activation is permanent, this provides a cumulative assay of exposure to Cre with good sensitivity to low concentrations. Finally, these new assays will be used to investigate whether transport is altered in AD mouse models.

概括 假设以对流流的形式的间质流体运动有助于淀粉样蛋白β（Aβ）和废物 通过将细胞外蛋白载出大脑的清除。该系统的失败可能有助于 阿尔茨海默氏病（AD），但仍然是该理论的某些方面。例如，流入 脑实质的液体已充分记录，出口路径不太清楚。外源示踪剂和 脑蛋白从组织进入淋巴染仍然是一个空旷的问题。至少有两个想法。这 淋巴/淋巴运输机制预测动脉周围空间流体的流入 通过静脉周围空间的插座将平衡。动脉周围排水 （iPad）CAMP已经确定，细胞外染料和蛋白质在地下膜附近骨料聚集 动脉视频和假设该隔室为废物清除提供了管道。 iPad的想法 预测，沿着阿尔蒂尔（Artiolles 被认为可以进食淋巴细胞。研究这种运输方面存在实验挑战，因为 通常通过注入改变压力平衡的外源指标来跟踪流体运动，只能是 用于急性测量。为了克服这些障碍，新的基因工程分泌的示踪剂 （gests）哪些模仿内源性蛋白质的产生，并不因注射的伪影而变得复杂 被原型。显微注射的腺相关病毒（AAV）向量驱动神经元中的gest表达 在少量的大脑中。使用荧光成像在内的分泌荧光蛋白的分布 组织切片和插入成像均可用于绘制传输。因为这个示踪剂是由 在注射病毒载体，大脑中的压力和流体流量后很长时间，神经元不会受到干扰。在 此外，神经元还将诸如Aβ之类的蛋白质分泌，因此Gest分布将复制天然 蛋白质转运而不是外源注射示踪剂的运动。虽然该策略表现出了希望，但 需要新的功能来解决有关废物蛋白流体运输的具体问题。第一的， 大脑中的间质流体流速需要在几分钟的尺度上更快地分辨率。光活化 提出了gest的版本，以便可以“突出显示”示踪剂，并在其移动时进行跟踪 脑组织。运动的方向和速度可以支持或反驳流量介导的不同理论 废物清除。其次，无法检测到更快的流量或低浓度区域的gest，例如 淋巴技术。为了检测分泌蛋白所采取的路径，开发了一种新的分泌的细胞 - 佩里CRE。 这将用于floxped的记者动物，以打开遇到流体的细胞中的报告基因 携带CRE。因为基因激活是永久的，所以这提供了对暴露的累积评估 对低浓度的敏感性。最后，这些新测定将用于调查是否 在AD鼠标模型中，传输发生了变化。